PT - JOURNAL ARTICLE
AU - SADLISH, Heather
AU - WILLIAMS, Frederick M.R.
AU - FLINTOFF, Wayne F.
TI - Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function
AID - 10.1042/bj20011361
DP - 2002 Jun 15
TA - Biochemical Journal
PG - 777--786
VI - 364
IP - 3
4099 - http://www.biochemj.org/content/364/3/777.short
4100 - http://www.biochemj.org/content/364/3/777.full
SO - Biochem J2002 Jun 15; 364
AB - The reduced folate carrier (RFC) protein has a secondary structure consistent with the predicted 12 transmembrane (TM) domains, intracellular N- and C-termini and a large cytoplasmic loop between TM6 and TM7. In the present study, the role of the cytoplasmic domains in substrate transport and protein biogenesis were examined using an array of hamster RFC deletion mutants fused to enhanced green fluorescent protein and expressed in Chinese hamster ovary cells. The N- and C-terminal tails were removed both individually and together, or the large cytoplasmic loop was modified such that the domain size and role of conserved sequences could be examined. The loss of the N- or C-terminal tails did not appear to significantly disrupt protein function, although both termini appeared to have a role in the efficiency with which molecules exited the endoplasmic reticulum to localize at the plasma membrane. There appeared to be both size and sequence requirements for the intracellular loop, which are able to drastically affect protein stability and function unless met. Furthermore, there might be an indirect role for the loop in substrate translocation, since even moderate changes significantly reduced the Vmax for methotrexate transport. Although these cytoplasmic domains do not appear to be absolutely essential for substrate transport, each one is important for biogenesis and localization.